Figure 7. The physical and functional association between Dcp1a and ARE-containing mRNA.

(A) Interaction between Dcp2 and Dcp1a in the extracts of 3T3-L1 cells. 70% confluent 3T3-L1 cells were transiently transfected with control vector or Flag-Dcp2 plasmids. After two days, the cells were untreated or treated with FMDI for 1 h and cell extracts were isolated for IP with anti-Flag M2 agarose beads. The precipitated protein complexes were western blotted with anti-Dcp1a, anti-Flag, anti-p-Ser315, and anti-tubulin. Input is 10% protein amount. (B) RNA pull-down. Cytoplasmic extracts from 3T3-L1 control cells or induced to differentiation for 0.5, 1 and 2 h were incubated with in vitro transcribed biotinylated MKP-1 ARE and control 18S RNA. The protein and biotinylated RNA complexes were recovered by addition of Streptavidin Sepharose. The brought-down complexes were resolved by gel electrophoresis followed by western blotting with anti-Dcp1a and anti-Brf1 antibodies. (C) Kockdown of Dcp1a in 3T3-L1 cells. The knockdown efficiency of Dcp1a was determined by western blotting with anti-Dcp1a and anti-tubulin. shLuc is a negative control. (D) Analysis of MKP-1 mRNA stability. The shLuc control cells, Dcp1a knockdown cells, and the Dcp1a knockdown cells overexpressed with wild-type (WT) or S315A/S319A (AA) or S315D/S319D (DD) GFP-tagged Dcp1a were induced by FMDI for 1 h, and then 10 µg/ml of actinomycin D was added for 0, 20 and 40 min. The RNA was isolated for quantitative PCR analysis by using primers of MKP-1 and actin. The graph displayed the MKP-1 mRNA remaining levels in the different treated cells as indicated. (E) The Dcp1a expression levels were detected by western blotting with anti-GFP (upper) and anti-Dcp1a (middle). The lower panel showed the tubulin expression levels as an internal control.